Method and reagent for determining total cholesterol in blood serum

ABSTRACT

In a method for the determination of total cholesterol in blood serum, plasma or other cell free body fluid the steps of adding a reagent comprising a mixture of ferric acetate (Fe(C2H3O2)3) and uranium acetate (UO2(C2H3O2)3) to the fluid whereby the total cholesterol content thereof is solubilized and those chromogens which interfere with the determination are precipitated and separating the liquid phase containing solubilized cholesterol from the precipitate. The cholesterol content is then determined quantitatively, preferably colorimetrically, employing as a color-developing reagent a mixture of ferrous sulfate and sulfuric acid. The invention also includes the ferric acetateuranium acetate reagent and the ferrous sulfate-sulfuric acid color reagent.

United States Patent Primary Examiner-Joseph Scovronek Attorney-Stowe &Stowell ABSTRACT: In a method for the determination of total cholesterolin blood serum, plasma or other cell free body fluid the steps of addinga reagent comprising a mixture of fer ric acetate [Fe(C H -,O anduranium acetate [UO (C 11 0 to the fluid whereby the total cholesterolcontent thereof is solubilized and those chromogens which interfere withthe determination are precipitated and separating the liquid phasecontaining solubilized cholesterol from the precipitate. The cholesterolcontent is then determined quantitatively, preferably colorimetrically,employing as a colordeveloping reagent a mixture of ferrous sulfate andsulfuric acid. The invention also includes the ferric acetate-uraniumacetate reagent and the ferrous sulfate-sulfuric acid color reagent.

[72] Inventors Amritlal C. Parekh;

David H. Jung, both of Indianapolis, Ind. [21] Appl. No. 8,954 [22]Filed Feb. 5, 1970 [45] Patented Oct. 26, 1971 [73] Assignee ResearchCorporation New York, N.Y.

[54] METHOD AND REAGENT FOR DETERMINING TOTAL CHOLESTEROL IN BLOOD SERUM10 Claims, 4 Drawing Figs.

[52] U.S. Cl 23/230 B, 23/312 A, 252/363.5, 252/364, 252/408, 260/397.25[5 1] Int. Cl G0ln 33/16 [50] Field of Search 23/230 B, 259, 230, 312 A;252/408, 363.5, 364; 260/397.25

[56] References Cited UNITED STATES PATENTS 1,722,435 7/1929 Leiboff23/230 B X 2,359,128 9/1944 Leibofi' 23/259 600 LLI (I D D 8 o I! 500 D.

J 1 ll] l m 1 3 400 E o I 9 i g 300 2 j 5 1 i l 1 o I a I l LT, 200 i .2m l O I U l 5 I00 o tz 1 1 O l l TOTALCHOLESTEROL (mg/IOOmUUTILIZlNGFERRIC ACETATE URANIUM ACETATE REAGENT METHOD AND REAGENT FORDETERMINING TOTAL CHOLESTEROL IN BLOOD SERUM BACKGROUND OF THE INVENTIONThe present invention relates to a reagent and method for quantitativelydetermining the amount of total cholesterol in body fluids, preferablycolorimetrically.

Cholesterol exists in substantially all plant and animal cells either inthe free form or as an ester. It ofien exists in admixture with one ormore of its derivatives such as dehydrocholesterol,7-dehydrocholesterol, etc. Cholesterol is an essential ingredient inhuman blood and is present in constant amounts therein in normal,healthy human beings. The cholesterol level in human blood is usuallymeasured as total cholesterol" which includes the sum of the freecholesterol and its derivatives. In human blood, these derivativespredominantly comprise esters thereof with the fatty acids contained inhuman blood.

Recent medical research has unearthed evidence which directly equatesthe total cholesterol content of blood with certain maladies. Forexample, the total cholesterol count has been found to be unusually highin the blood of human beings suffering from diabetes, certain diseasesof the liver, familial hypercholesterolemia, alcoholism, syphilis,nephritis and other maladies. Most importantly, a high blood cholesterolcontent has been found to have a direct bearing on the incidence ofatherosclerosis and other vascular difiiculties.

There have been recently introduced several methods and reagents forquantitatively determining the cholesterol content of human blood. Themost successful of these methods involve the colorimetric determinationof cholesterol following the addition to serum samples of a reagentadapted to react with cholesterol and its derivatives and form a color.The intensity of this color may be measured in a suitable apparatus suchas a spectrophotometer, etc. The amount of cholesterol may then becalculated by comparing this measured intensity with that of a knownstandard.

Inasmuch as cholesterol and its derivatives are highly insoluble, it isnecessary to add a reagent or solvent to the blood serum to solubilizethe cholesterol prior to reaction with the color-developing reagent. Inthe past it has been suggested to employ various materials to solubilizecholesterol. All of the previously employed solubilizing agents sufferfrom serious disadvantages, however. Although several of the proposedmethods effectively solubilize cholesterol, they also solubilize othermaterials in the serum which interfere with the colorimetricdetermination, e.g., proteins, bilirubin, lipids, etc. Inasmuch as thesematerials are color-developing during the conventionally employedcolorimetric determinations they will obviously seriously interfere withthe obtention of a true reading of the intensity of color or opticaldensity attributable to cholesterol. Furthermore, since the content ofthese materials in human blood varies from person to person, it isvirtually impossible to apply a correction factor in the colorimetricanalysis.

The standard Abel] method, while it does not suffer from theabove-described disadvantage, is extremely complicated and timeconsuming in order to avoid the solubilization of interferingchromogens.

The prior art is also faced with a serious problem in that to date thereagents employed to develop color by reaction with cholesterol and itsderivatives have been found to be unsatisfactory. The most commonly usedreagents are the Liebermann-Burchard reagent and a FeCL -I-LSO mixture.The former comprises a mixture of acetic anhydride and sulfuric acidwhich develops a green color. The colored reaction product is, however,unstable and requires measurement after the lapse of precise timeintervals.

The FeCI -ILSO reagent forms a more stable colored reaction product butsuffers from the disadvantage that RC1 is evolved during thecolor-forming reaction thereby causing variations in the reactiontemperature. The reagent also contributes a yellow color both in thesample under analysis and in the blank or standard with which it iscolorimetrically compared thereby interfering with measurement of theoptical density.

SUMMARY OF THE INVENTION By the present invention there is provided amethod for the quantitative determination of cholesterol in body fluidswhich is relatively simple and easy to carry out utilizing onlymicroquantities of the body fluid, yet is extremely accurate and givesresults which are highly reproducible.

By the present invention there is also provided a reagent for thecolorimetric determination of cholesterol in body fluids whichselectively solubilizes cholesterol and does not solubilize thosechromogens which interfere with the quantitative determination.

The reagent of the present invention comprises a mixture of ferricacetate and a heavy metal acetate, preferably, uranium acetate [Fe(C,I-IO, and UO,(C,I-I,O,,]. The reagent is employed in the method of theinvention as a lower aliphatic carboxylic acid, preferably, acetic acidsolution. This reagent possesses the unique property of being able tosolubilize the total cholesterol content of blood serum whilesimultaneously precipitating those chromogens which interfere with thequantitative determination such as proteins, lipids, bilirubin, etc.

The method according to the present invention comprises intimatelyadmixing the above-defined reagent with a sample of body fluid,separating at least a portion of the liquid phase containing thesolubilized cholesterol from the precipitated chromogens andquantitatively analyzing the separated liquid phase for its cholesterolcontent. Since the interfering chromogens have been substantiallyentirely eliminated from the liquid phase there is virtually nointerference with the analysis. The cholesterol is preferably determinedcolorimetrically.

The reagent employed to develop the color in the separated liquid phaseis preferably a mixture comprising sulfuric acid and ferrous sulfate.When added to the above-described separated liquid phase, an intensepurple color is developed. Moreover, no side products having colorswhich interfere with the colorimetric analysis are produced.

As will be apparent to those skilled in the art, the abovedescribedmethod is extremely simple and capable of being carried out without thenecessity for extensive training of the operator.

DETAILED DESCRIPTION OF THE INVENTION The method and reagent of theinvention are adapted for the solubilization and determination of thetotal cholesterol in human body fluids, i.e., the sum total of the freecholesterol and cholesterol derivatives such as dehydrocholesterol, 7-dehydrocholesterol, cholesterol esters, etc. It is an advantage of themethod and reagent of the invention that the quantitative determinationof total cholesterol is not affected to any significant extent by thepresence in the body fluid of other steroids, even in amounts exceedingtheir physiological concentrations.

The invention is applicable for the determination of total cholesterolin any cell-free body fluid. Suitable such fluids include blood serum,blood plasma, cell-free tissue homogenizates, etc.

As described above, the reagent of the present invention comprises amixture of ferric acetate and uranium acetate [Fe(C,I-IO and UO (C H O,)The reagent is a mixed salt and is preferably employed in the method ofthe invention as a lower aliphatic carboxylic acid, preferably aceticacid solution. The reagent is conveniently prepared by dissolving ferrichydroxide in acetic acid and then adding uranium acetate. The ratio offerric acetate to uranium acetate in the mixed salt may vary from about4:1 to about 4.5:1 by weight. Generally, the

The ferric acetate-uranium acetate reagent of the invention may beutilized to solubilize cholesterol and precipitate the interferingchromogens in the fluid sample analyzed by any quantitative method whichdepends upon the determination of solubilized cholesterol. Preferably,the reagent is utilized to solubilize the cholesterol for colorimetricanalysis wherein the solubilized cholesterol is reacted with acolor-forming reagent and the resulting color is measured, i.e., with aspectrophotometer. The reagent is preferably employed in a method,however, which depends upon the reaction of the solubilized cholesteroland a color-developing reagent comprising a mixture of sulfuric acid andferrous sulfate. As noted above, the utilization of an H SQ FeSO reagentdoes not result in the production of undesirable color-forming sideproducts or in variations in the reaction temperature. FeSO isdifficulty soluble in concentrated H 80 and is therefore preferablyfirst dissolved in glacial acetic acid prior to admixture with thesulfuric acid.

The method of the invention comprises adding a sufficient amount of theferric acetate-uranium acetate reagent to the blood serum to be analyzedto solubilize the total cholesterol content thereof and precipitate allof the interfering chromogens contained therein, i.e., proteins, lipids,bilirubin.

Generally, an amount of the reagent is added to the body fluid sample,either in solid or solution form, sufiicient to provide a sample toreagent ratio, by weight, of from about 1:50 to about 1:300, preferablyfrom about 1:100 to about 1:250, is sufficient to solubilize thecholesterol and precipitate the interfering chromogens. A sample toreagent ratio of about 1:200 is most preferable.

The ferric acetate-uranium acetate reagent and body fluid sample areintimately admixed and the mixture allowed to stand for a sufficientperiod of time to ensure complete solubilization of all of thecholesterol contained in the sample and to ensure complete precipitationof all of the interfering chromogens contained therein. The amount oftime required is obviously dependent upon a variety of factors,including the size of the sample, amount of reagent employed, thecholesterol and other chromogen content of the sample, etc. Generally,however, where a sample size of from about 0.001 to about 0.1 ml. is tobe analyzed and sufficient reagent is employed to provide a sample toreagent ratio by weight of from about 1:50 to about 1:300, a period oftime for precipitation of interfering chromogens and proteins of fromabout 5 to about 15 minutes, preferably from about 5 to about 7 minutes,is usually sufficient.

Following complete solubilization of the cholesterol and precipitationof the interfering chromogens at least a portion of the liquid phase ofthe body fluid sample is separated from the precipitate. This is mostconveniently accomplished by centrifuging the sample to segregate theliquid phase and the precipitated solids. A portion of the supernatantliquid phase is then drawn off and transferred to a suitable containerfor colorimetric or other quantitative analysis.

The amount of liquid phase drawn off is not critical and will dependupon the particular method contemplated for analysis. Where color for acolorimetric analysis is developed employing the preferred FeSO -H,SOreagent of the present invention, a portion of the liquid phaseamounting to from about 0.01 to about 5 ml. is generally sufi'lcient forsubsequent colorimetric analysis.

As noted above, any conventional colorimetric method may be employed toanalyze the body fluid sample. To avoid variations in reactiontemperature and the production of side products during the color-formingreaction which interfere with the colorimetric determination, however,it is preferred to employ a colorimetric method which depends upon thecolor formed by reaction of the solubilized cholesterol with a FeSO,-H,SO, reagent. It has been found that the FeS -H,SO reagentproduces a color, the measurement of which colorimetrically ensures acorrect analysis.

The reagent preferably comprises a substantially anhydrous mixture ofsulfuric acid and ferrous sulfate. The ratio of ferrous sulfate tosulfuric acid, by weight, may range from about 110,000 to aboutl:30,000, preferably from about 1:15.000 to about 120,000. Mostpreferably a mixture having a ferrous sulfate to sulfuric acid ratio ofabout 1218,000 is employed. Optionally, the reagent may contain a smallamount of acetic acid which is employed to aid in the solubilization ofthe FeSO In order to develop a sufficiently intense color to enable aprecise colorimetric determination of the total cholesterol in the bodyfluid sample it is generally necessary to add sufficient reagent to theliquid phase containing solubilized cholesterol to provide a reagent:liquid phase ratio, by weight, of from about 1:2to about 4: 1,preferably from about 1:1 to about 3:1. Most preferably, an amount ofreagent is employed which provides a reagent: liquid phase ratio ofabout 1.23:1.

Upon addition of the FeSO -H SO, reagent to the liquidphase aliquotcontaining solubilized cholesterol an intense purple color is developed.The sample is allowed to stand for a period of time sufficient to permitmaximum color development, generally, from about 5 to about 60 minutes.The sample is then analyzed colorimetrically; most conveniently bymeasuring the optical density of the sample in a spectrophotometeragainst a blank containing an amount of ferric acetate-uranium acetatesolution equal to the size of the liquid-phase portion analyzed and anamount of FeSO,-l-1,SO, reagent identical to that employed to developthe color therein. It is to be understood, however, that anycolorimetric method may be employed to analyze the sample.

The invention will be illustrated by the following nonlimiting example.

EXAMPLE 1 a. Preparation of ferric acetate-uranium acetate reagent 0.5gm. of FeCl;, 6H O is converted to Fe(Ol-l by admixing concentratedammonia therewith. The Fe(OH is filtered and washed with water anddried. The Fe(Ol-l), is then dissolved in glacial acetic acid and thevolume of the solution made up to 1 liter with further glacial aceticacid. mg. of uranium acetate [UO (C H O,) '2l-l O)] is then dissolved inthe solution and the resulting solution allowed to stand overnight.

b. Preparation of FeSO -H SO reagent To 0.1 gm. of anhydrous ferroussulfate in a flask is added 100 ml. of glacial acetic acid whileswirling and then 100 ml. of concentrated sulfuric acid is added. Afterdissolution of the ferrous sulfate and cooling to room temperature thevolume of the solution is made up to 1 liter with concentrated sulfuricacid.

c. Determination of cholesterol 10 ml. of the ferric acetate-uraniumacetate reagent prepared according to the above-described process areadded to a l6Xl 25 mm. tube provided with a screw cap. 50 pl. of a bloodserum sample is added to the tube and thoroughly admixed with thereagent. The tube is allowed to stand undisturbed for 5 minutes. Aprecipitate is observed to have been formed in the mixture. The mixtureis centrifuged for 5 minutes.

A 3-ml. aliquot of the supernatant liquid phase is drawn off with apipette and transferred to another l6Xl25-mm. tube, unscratched,provided with a screw cap, which is adapted for use as a cuvette.

2 ml. of the FeSO H SO reagent prepared according to the above-describedprocess is added to the aliquot. The tube is tightly capped and thecontents thoroughly admixed. The tube is allowed to stand for 20 minutesto permit maximum color development. The purple color of reaction ismeasured in a spectrophotometer at 560 my. against a blank consisting of3 ml. of ferric acetate-uranium acetate solution and 2 ml. of FeSO,-HSO, reagent.

EXAMPLE 2 total cholesterol in human blood serum, the procedure ofexample l is followed in analyzing six different icteric blood sera. Thesame six sera are also analyzed according to the procedure set forth inexample 1 but employing as the solubilizing reagent, ferric acetate,prepared according to example in with the exception that no uraniumacetate is added to the solution. The total cholesterol is measured inmg./ 100 ml. by comparing the measured optical densities with those ofstandard cholesterol solutions which were analyzed according to the sameprocedure. The same six sera are also analyzed according to thewell-known Abell procedure which is commonly accepted as the mostaccurate method, although difficult, tedious and time consuming, forquantitatively determining cholesterol. The Abell procedure is describedin J. Biol. Chem., Vol. l95, page 357 (1952) and in Standard Methods inClinical Chemistry, Vol. 2, page 26 (1958).

The results are set forth in table 1.

TABLE 1 Mg Cholesterol/I00 ml Serum Abel] Fe(C;HaO )1 No. ProcedureUO2(C2H301)1 Fe(C=H;O2)3

As is apparent from table 1, the results obtained according to themethod of the present invention compare favorably with the highlyaccurate results obtained according to the Abell procedure. Whenemploying the ferric acetate reagent, however, which solubilizes boththe total cholesterol and interfering chromogens, the results obtainedare higher in each instance, thus demonstrating the effectiveness of thereagent of the invention in eliminating the interfering chromogens.

EXAMPLE 3 TABLE 2 mg. Cholesterol/100 ml.

Serum Abel] No. Procedure H,SO,-FeSO, Cone. H,SO

l. 375 375 400 2. 307 305 326 3. I68 H0 H6 4. I27 128 132 5. I72 I73 I806. I26 I29 135 As is apparent from table 2, the results obtained whenfollowing the method of the invention and employing the FeSO.,- H 80,color-developing reagent compare favorably with those obtained followingthe Abel] procedure. When employing only H,SO,, however, the resultsobtained are consistently higher.

EXAMPLE 4 The procedure of example 1 is repeated employing samples froma serum pool containing 82 mg./ 100 ml. cholesterol to which were addedvarying amounts of cholesterol. The results are set forth in table 3.

TABLE 3 Cholesterol Total Cholesterol Added Cholesterol Content RecoveryPercent (mgJlOO ml.) (mg/I00 ml.) (mg/I00 ml.) Recovered 100 I82 I82l00.0

EXAMPLE 5 The procedure of example 1 is followed utilizing ferricacetate reagents having different concentrations in acetic acid. A highcholesterol content serum was employed (400 mg./ 100 ml.). The resultsare set forth in table 4. As is apparent, a concentration of ferricacetate of about 5.6 mEq/l. is sufficient for cholesterol solubilizationand subsequent color development. By employing ferric acetate as thesolubilizing component of the reagent rather than the conventionallyemployed ferric chloride, the objectionable evolution of HCl occasionedby the use of the latter is avoided.

The procedure of example 1 is followed with the exception that theamount of l=eSO,,-l'l-,.S0 reagent is varied over six separate runs. Theresults are set forth in table 5.

TABLE 5 Volume of H,SO,-FeSO, Absorbance These results indicate that theoptimum ratio of ferric acetate-uranium acetate reagent to ferroussulfate-sulfuric acid reagent is about 3:2.

EXAMPLE 7 The procedure of example 1 is followed in analyzing two serumsamples. In one analysis the color-developing reagent employed is FeSO-I-I,SO.,. In the second analysis, an equivalent volume of concentratedl-I SO is employed. The optical densities are measured at different timeintervals. The results are set forth in table 6.

As is apparent, the color developed employing the FeSO H 80 reagent ismore intense and is more stable than that produced when employing H 80,alone. When using the latter, the color produced is less intense thanwhen using the former and it fades on standing.

EXAMPLE 8 The procedure of example 1 is followed in analyzing a standardcholesterol sample (200 mg./ 100 ml.) and a serum sample. The opticaldensities are measured at various wavelengths. The optical densities ofthe blank are measured against water. The results are set forth in table7.

As is apparent, the absorbance of the blank is ideally low,demonstrating the lack of contribution of interfering colorproducingside reaction products by the method of the invention. Moreover, as isapparent from the table the standard and serum samples evidenceidentical peaks at about 560 mu.

EXAMPLE 9 The procedure of example 1 is followed in setting up acalibration curve. Various cholesterol standards and Serachol solutions(commercial cholesterol standard solutions manufactured byWamer-Chilcott) of various concentrations are analyzed and theabsorbancies plotted against the known concentrations. The thus-obtainedcalibration curve is set forth in FIG. 1.

As is apparent, the calibration curve is absolutely linear, even up tol,000 mg./ 100 ml., thus evidencing the unusually extensive analyticalrange of the method and reagent of the invention.

EXAMPLE 10 The procedure of example 1 is followed in analyzing variousrandomly selected serum samples. The same samples are also analyzedaccording to the Abell procedure. The results are plotted against eachother in terms of mg. cholesterol/ ml. The resulting scattergram isdepicted in FIG. 2.

As is apparent the linearity of the scattergram indicates that theresults obtained according to the method of the invention are highlyaccurate over a wide cholesterol concentration range.

EXAMPLE 1 l The procedure of example 1 is followed in analyzing varioussamples of both icteric sera and lipemic sera.

The same samples are analyzed according to the Abell procedure. Theresults (mg. cholesterol/I00 ml.) are plotted in the scattergram setforth in FIG. 3.

Again, the results demonstrate the effectiveness of the reagent andmethod of the invention in eliminating the interference of chromogenssuch as bilirubin, proteins, lipids, etc. with the quantitativedetermination of cholesterol by a colorimetric method.

EXAMPLE 12 The procedure of example 1 is followed in analyzing serumsamples which have been preliminarily extracted according to the methodof Abell, i.e., 0.5 ml. of Abells extract is evaporated to dryness,reconstituted with 3 ml. of the ferric acetate-uranium acetate reagentsolution and admixed with 2 ml. of the FeSO -l-I,S0 reagent.

The procedure of Abell is followed in analyzing the same samples. Theresults are depicted in the scattergram set forth in FIG. 4. As isapparent therefrom, the method and reagent of the invention may beemployed equally well in analyzing serum samples by whatever methodutilized to extract the cholesterol therefrom.

It is readily apparent from the foregoing examples that the method andreagent of the invention enable precise quantitative determinations ofcholesterol with relative ease and simplicity of operation.

We claim:

1. A method for the colorimetric determination of total cholesterol in acell cell-free body fluid sample comprising:

a. intimately admixing said sample and an acetic acid solution of amixed salt comprising ferric acetate and uranium acetate and allowingthe mixture to stand for a period of time sufficient for the cholesterolin said sample to be solubilized and the chromogens contained in saidsample which interfere with said colorimetric determination to beprecipitated,

b. separating at least a portion of the liquid phase containingsolubilized cholesterol from said precipitate, and

c. analyzing said liquid phase colorimetrically by measuring the opticaldensity thereof after the addition thereto of a color-developingreagent.

2. A method according to claim 1 wherein said colordeveloping reagent isa mixture of ferrous sulfate and sulfuric acid.

3. A method according to claim 1 wherein the amount of total cholesterolin said sample is determined by comparing the optical density measuredin step c with that of a standard.

4. A method according to claim 1 wherein the weight ratio of ferricacetate to uranium acetate in said mixed salt is from about 4:1 to about4.521.

5. A method according to claim 4 wherein said acetic acid solutioncontains from about 0.05 to about 0.06 percent by weight of said mixedsalt.

6. A method according to claim 4 wherein the weight ratio of said sampleto said reagent is from about 1:50 to about 1:300.

7. A method according to claim 2 wherein the ratio of ferrous sulfate tosulfuric acid by weight in said color-developing reagent is from aboutl:l0,000 to about l:30,000.

8. A method according to claim 2 wherein the ratio of saidcolor-developing reagent to said liquid phase containing saidsolubilized cholesterol, by weight, is from about 1:2 to about 4:1.

9. A method according to claim 2 wherein said colordeveloping reagent issubstantially anhydrous.

10. A method for the quantitative colorimetric determination of totalcholesterol in a cell-free body fluid sample comprising:

a. intimately admixing said sample with about 200 parts by weight basedon the weight of said sample of an acetic acid solution containing about0.055 percent, by weight, of a mixed salt comprising ferric acetate anduranium acetate, the weight ratio of ferric acetate to uranium acetatebeing from about 4:1 to about 4.511,

b. allowing the mixture to stand for from about 5 to about min. wherebythe total cholesterol in said sample is separating 3 ml. of said liquidphase from said segregated mixture,

. adding to said separated liquid phase 2 ml. of a reagent comprising amixture of sulfuric acid and ferrous sulfate wherein the ratio offerrous sulfate to sulfuric acid, by

weight, is about 1218,000 whereby a purple color of reaction isdeveloped,

analyzing said liquid phase colorimetrically by measuring the opticaldensity thereof, and

determining the total cholesterol content of said sample by comparingsaid measured optical density with that of a standard.

2. A method according to claim 1 wherein said color-developing reagentis a mixture of ferrous sulfate and sulfuric acid.
 3. A method accordingto claim 1 wherein the amount of total cholesterol in said sample isdetermined by comparing the optical density measured in step c with thatof a standard.
 4. A method according to claim 1 wherein the weight ratioof ferric acetate to uranium acetate in said mixed salt is from about4:1 to about 4.5:1.
 5. A method according to claim 4 wherein said aceticacid solution contains from about 0.05 to about 0.06 percent by weightof said mixed salt.
 6. A method according to claim 4 wherein the weightratio of said sample to said reagent is from about 1:50 to about 1:300.7. A method according to claim 2 wherein the ratio of ferrous sulfate tosulfuric acid by weight in said color-developing reagent is from about1:10,000 to about 1:30,000.
 8. A method according to claim 2 wherein theratio of said color-developing reagent to said liquid phase containingsaid solubilized cholesterol, by weight, is from about 1:2 to about 4:9. A method according to claim 2 wherein said color-developing reagentis substantially anhydrous.
 10. A method for the quantitativecolorimetric determination of total cholesterol in a cell-free bodyfluid sample comprising: a. intimately admixing said sample with about200 parts by weight based on the weight of said sample of an acetic acidsolution containing about 0.055 percent, by weight, of a mixed saltcomprising ferric acetate and uranium acetate, the weight ratio offerric acetate to uranium acetate being from about 4:1 to about 4.5:1,b. allowing the mixture to stand for from about 5 to about 10 min.whereby the total cholesterol in said sample is solubilized and thechromogens contained in said sample which interfere with saidcolorimetric determination are precipitated, c. centrifuging saidmixture to segregate said precipitate from a liquid phase containingsolubilized total cholesterol, d. separating 3 ml. of said liquid phasefrom said segregated mixture, e. adding to said separated liquid phase 2ml. of a reagent comprising a mixture of sulfuric acid and ferroussulfate wherein the ratio of ferrous sulfate to sulfuric acid, byweight, is about 1:18,000 whereby a purple color of reaction isdeveloped, f. analyzing said liquid phase colorimetrically by measuringthe optical density thereof, and g. determining the total cholesterolcontent of said sample by comparing said measured optical density withthat of a standard.